For plasmids: From our experience, it is CRITICALLY important that the DNA template be clean and free of contaminants! The success of your sequencing reaction depends on many things, most importantly on the purity and cleanliness of your DNA. We recommend Qiagen, BioRad, and Clonetech kits for plasmid purification. Most importantly, whichever prep you choose, do not resuspend in TE. The EDTA in the TE acts as a chelating agent capturing the Mg2+ needed as a co-factor for the polymerase in our cycle sequencing reaction. This will inhibit your reaction and could cause it to fail completely. We recommend resuspending your DNA in ddH20. Qiagen EB buffer or Tris 8.0 are also fine.
For PCR products: The DNA should be purified using a reputable PCR purification method such as QIAquickTM or AMPureTM to remove excess primers and dNTP’s after your PCR cycling. Please run your PCR products out on a gel to be sure that you do not have multiple products. Elute your PCR products with ddH20 or Tris 8.0 buffer, no TE here either.
Click here for DNA Concentration Guidelines
A word about DNA concentrations...we recommend that you not only UV spec your samples, but also double check them on a gel to check concentration.
Here is what we need from you....
Template DNA: We need 6µL of template DNA per reaction placed in a well labeled 0.5mL tube. If the template will be used for more than one reaction, no need for separate tubes, you may simply increase the volume in that tube based on how many reactions are to be performed with that template. For example, if you are running 2 reactions with the same template, we would need a total of 12µL of template DNA in the tube.
Please be sure the names on the tops of your tubes correspond to what is on your request form.
Primers: Please supply us with 4µL of primer per reaction at 3µM, or 20ng/µL, in a separate tube from the template DNA unless you are using our facility supplied universal primers which are free of charge to you. Similar to the template DNA, If the primer will be used for more than one reaction, no need for separate tubes, you may simply increase the volume in that tube based on how many reactions are to be performed with that primer. For example, if you are running 4 reactions with the same primer, we would need a total of 16µL of primer in the tube. Again please be sure the names on the tops of your tubes correspond to what is on your request form.
For larger orders please note: If you will be taking advantage of our bulk discount prices and submitting 48 or more samples at once, please submit your samples in a v-bottom 96-well PCR plate. Be sure you aliquot your samples according to our directions. Samples1-8 should be in wells A1-H1, samples 9-16 should be in wells A2-H2, etc. (click here for diagram). For your convenience, you will notice that these well numbers correspond to the well numbers on our Excel spreadsheets. In an effort to avoid concentration imbalances that can lead to poor quality sequencing results, we ask that you do not combine template and primer in the same well, but rather submit them separately. Primers can also be submitted in tubes. Be sure to label plate and primer/s clearly with your name and order number and cover the plate with an adhesive seal (plastic or foil). Please do not use strip caps as contamination can be a problem.
Disclaimer for multiple plate orders: Please note that if you have a large order with multiple plates and you leave excessive sporatic blanks in your plate and the Excel request form, you will be charged for the empty wells. It is difficult and error prone to fill in those blanks on our gels with other customers' orders. Thank you!